In this report period, we carried on our long-standing research line, development of anti human immunodeficiency virus (HIV) drugs. We particularly focused two major research areas relating to the treatment of HIV-1 infection. (1) We identified several groups of novel agents active against a wide spectrum of HIV including multi-drug-resistant HIV variants. (2) We also attempted to elucidate the mechanism(s) of the emergence of HIV-1 variants resistant to multi nucleoside reverse trasncriptase inhibitors (HIV-1MDR). We designed, synthesized, and identified UIC-94017 (TMC114), a novel nonpeptidic HIV-1 protease inhibitor (PI) containing a 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) and a sulfonamide isostere, which is extremely potent against laboratory HIV-1 strains and primary clinical isolates (IC50: 0.003 mM; IC90: 0.009 mM) with minimal cytotoxicity (CC50: 74 mM in CD4+ MT-2 cells). UIC-94017 blocked the infectivity and replication of each of HIV-1NL4-3 variants exposed to and selected by up to 5 mM concentration of saquinavir, indinavir, nelfinavir, or ritonavir (IC50: 0.003 - 0.029 mM), although it was less active to HIV-1NL4-3 selected by amprenavir (IC50: 0.22 mM). UIC-94017 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents. Structural analyses revealed that the close contact of UIC-94017 with the main chains of the protease active site amino acids (Asp-29 and Asp-30) is important for its potency and wide-spectrum activity against multi-PI-resistant HIV-1 variants. Considering the favorable pharmacokinetics of UIC-94017 when administered with ritonavir, the present data warrant that UIC-94017 be further developed as a potential therapeutic agent for treatment of infection with primary and multi-PI-resistant HIV-1. It is of note that a patent application for the identification and characterization of UIC 94017 and its analogs have been filed and licensed to Johnson and Johnson/Tibotec and UIC 94017 is currently undergoing phase IIa clinical trials in Europe. We previously reported that a series of novel low molecular weight spirodiketopiperazine derivatives including a prototypic E913 (Mr. 484). In this report period, we designed, synthesized, and identified a far more potent CCR5 inhibitor, designated AK602. AK602 exhibited a high affinity to CCR5 (KD values of 3 nM), exerted potent activity against a wide spectrum of laboratory and primary R5 HIV-1 isolates including multi-drug resistant HIV-1MDR (IC50 values of 0.2-0.6 nM), and potently blocked HIV-1-gp120/CCR5 binding. AK602, despite its much greater anti-HIV-1 activity than other previously published CCR5 inhibitors including TAK779 and SCH-C, preserved RANTES and MIP-1b binding to CCR5+ cells and their functions, while TAK779 and Sch-C fully blocked the CC-chemokines/CCR5 interactions. When intraperitoneally administered twice daily to R5 HIV-1JRFL-infected hu-periphearl blood mononuclear cells-transplanted, IL-2Rg-chain-knocked-out, non-obese diabetes-SCID mice, AK602 blocked CD4+ cell decrease and suppressed plasma HIV viremia by 2 logs. Pharmacokinetic studies revealed favorable oral bioavailability in rodents. These data warrant further development of AK602 as a potential therapeutic for HIV-1 infection.We also investigated the mechanism by which the Q151M mutation in reverse transcriptase (RT) requiring a two base change (CAGATG) develops, which confers multi-dideoxynucleoside resistance on HIV-1 and attempted to elucidate the reason for the relatively lengthy period of time required for its emergence under therapy with multiple nucleoside RT inhibitors (NRTIs). We compared the fitness of various infectious clones including two putative intermediates (HIV-1Q151K(AAG) and HIV-1Q151L(CTG)) for HIV-1Q151M(ATG), in relation to their sensitivity to drugs. Propagation assays and competitive HIV-1 replication assays were employed to evaluate viral fitness of various clones in relation to drug sensitivity of such clones to zidovudine and didanosine. Steady-state kinetic constants of recombinant RTs were also determined. The data showed that HIV-1Q151M most likely develops through a poorly replicating HIV-1Q151L, however, it also possibly occurs through two concurrent base changes. The data explained the mechanism by which HIV-1Q151M emerges after long-term chemotherapy with NRTIs.The emergence of HIV variants resistant to multiple anti-HIV agents represents a major cause of "treatment failure"in those receiving antiviral therapy of AIDS. The virological significance of amino acid (AA) insertions near the Gag protein cleavage sites in multi-PI resistant HIV variants is not known. When we newly generated full-sized infectious molecular clones using HIV isolates obtained from heavily drug-experienced/therapy-failed AIDS patients, of such 6 full-sized infectious clones generated, 4 clones were found to have unique insertions (TGNS, SQVN, AQQA, SRPE, APP, and/or PTAPPA) near the p17/p24 and p1/p6 Gag cleavage sites in addition to the known multiple AA substitutions within the protease (PR). The competitive HIV replication assay proved that such Gag-inserts mostly compromised the replication of wild-type HIV, whereas the primary clones containing inserts replicated significantly better than those without the inserts. Western blot analyses revealed that the processing of Gag proteins by wild-type PR was impaired by the inserts, whereas that by mutant PR was substantially improved by the inserts but was greatly hindered without them.Thus we conclude that the inserts seen in the proximity of the Gag cleavage sites in highly multi PI resistant HIV variants restore the otherwise compromised enzymatic activity of mutant PR, enabling the multi-PI-resistant HIV variants to remain replication-competent, another newly identified mechanism of the acquisition of HIV drug resistance to protease inhibitors.